US11414648B2 - Methods and compositions for production of fallopian tube epithelium - Google Patents

Abstract

The fallopian tube epithelium (FTE) has been recognized as a site of origin of high-grade serous ovarian cancer (HGSC). However, absence of relevant in vitro human models that can recapitulate tissue-specific architecture has hindered understanding of FTE transformation and initiation of HGSC. Here, induced pluripotent stem cells (iPSCs) were used to establish a novel 3-dimensional (3D) human FTE organoid in vitro model containing the relevant cell types of the human fallopian tube as well as a luminal architecture that closely reflects the organization of fallopian tissues in vivo. Modulation of Wnt and nodal/activin signaling pathways provided iPSC differentiation into Müllerian cells and subsequent use of pro-Müllerian growth factors promoted FTE precursors. The expression of Müllerian markers verified correct cellular differentiation. An innovative 3D growth platform, which enabled the FTE organoid to self-organize into a convoluted luminal structure, permitted final differentiation to a FTE lineage. This powerful human-derived FTE organoid model can be used to study the earliest stages of HGSC development and to identify novel and specific biomarkers of early fallopian tube epithelial cell transformation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of International Application No. PCT/US2018/024198, filed Mar. 23, 2018, which designated the U.S. and that International Application was published under PCT Article 21(2) in English. This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/476,416, filed Mar. 24, 2017, the contents of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

Described herein are methods and compositions related to generation of fallopian tube cells and tissues from pluripotent stem cells.

BACKGROUND

High-grade serous carcinoma (HGSC), the most common subtype of epithelial ovarian cancer (˜70%), has the highest mortality rate among all gynecological cancers. HGSC has a poor prognosis due to a combination of factors including late stage at diagnosis and a high predilection for developing drug resistance. It is now accepted that the majority of HGSCs arise from the secretory cells of the fallopian tube epithelium (FTE). The discovery of in situ lesions in the fallopian tube fimbriae, namely serous tubal intraepithelial carcinoma (STIC), supports the concept of the FTE origin of serous “ovarian” carcinoma. STICs are part of the high grade serous carcinogenic sequence and are believed to include early p53 mutations resulting in a “53 signature” that develops into invasive serous carcinoma of the fallopian tube, and that secondarily involves the nearby ovarian surface. The discovery of an extra-ovarian origin of ovarian cancer is a fundamental advance toward improving early detection, prevention and treatment of this lethal disease. However, the lack of relevant in vitro human models that can recapitulate tissue-specific architecture and study early alterations has hindered the further understanding of the mechanisms of FTE transformation as well as the initiation and progression of HGSC. Thus, there is a great need in the art for FTE cells, organoids and associated methods thereof.

Stem cell derived 3D organoid cancer models are emerging as powerful tools for understanding pathological processes at the molecular and cellular level, and they will be a critical platform to study the earliest stages of ovarian cancer development, which is poorly understood. By generating accurate organotypic human models of early-stage disease, the Inventors expect to be able to identify novel and specific biomarkers of early fallopian tube epithelial cell transformation. In the long term, this project could lead an identification of molecular targets for chemoprevention and thus significant reduction in the number of deaths from ovarian cancer each year.

Described herein is an iPSC-derived cellular model of fallopian tube epithelium development with numerous potential practical applications, including the identification of early biomarkers to stage disease progression and better classification of genetic risk factors. Methods and compositions for generating iPSC-derived FTE, including 3D organoids, offers a promising platform for validation of new drugs and targets, and modeling resistance to therapy within the tumor micro-environment in rapid, cost efficient manner. By generating iPSC-derived 3D FTE organoid model with various inherited mutant sample, one can elucidate specific mutant dependent response to treatment and can thereby predict the drug sensitivity of individual patients based on their unique spectrum of genetic mutations, allowing for guidance in treatment decisions and optimizing clinical outcomes in cancer patients.

SUMMARY OF THE INVENTION

Described herein is a method for generating a fallopian tube epithelium (FTE), including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in the presence of at least one first growth factor and at least one induction molecule to generate mesoderm cells, further culturing the mesoderm cells in the presence of at least one second growth factor, at least one second induction molecule, and at least first one kinase inhibitor to generate intermediate mesoderm (IM) cells, additionally culturing the IM cells in the presence of at least one third growth factor, and at least one second kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one fourth growth factor into FTE. In other embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs). In other embodiments, the at least one first growth factor includes activin A, and the at least one induction molecule includes CHIR99021. In other embodiments, culturing the hPSCs includes about 2 days. In other embodiments, the at least one second growth factor includes BMP4, the at least one second induction molecule includes CHIR99021, and the at least one first kinase inhibitor includes ROCK inhibitor Y-27632. In other embodiments, further culturing the mesoderm cells includes about 2 days. In other embodiments, the at least one third growth factor includes WNT4 and at least one second kinase inhibitor includes ROCK inhibitor Y-27632. In other embodiments, additionally culturing the IM cells includes about 2 days. In other embodiments, the at least one fourth growth factor includes follistatin estrogen and progesterone. In other embodiments, the IM cells are organized as a spheroid. In other embodiments, spheroids are subsequently cultured in a matrix. In other embodiments, the FTE are organized as an organoid. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, mesoderm cells express one or more of: BRACHYURY and MIXL1. In other embodiments, IM cells express one or more of: PAX2, OSR1, GATA3, and WT1. In other embodiments, Mullerian epithelium cells express one or more of: WT1 and OVGP1. In other embodiments, Mullerian epithelium cells do not express one or more of: SIX2, FOXD1, CDX2 and NKX2-1.

Further described herein is a method of generating fallopian tube epithelium (FTE) organoids, providing a quantity of intermediate mesoderm (IM) cells, culturing the IM cells in a matrix and in the presence of at least one first growth factor, and at least first one kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one second growth factor into FTE organoids. In other embodiments, the at least one first growth factor includes WNT4 and at least one kinase inhibitor includes ROCK inhibitor Y-27632. In other embodiments, culturing the IM cells includes about 2 days. In other embodiments, the at least one second growth factor includes follistatin estrogen and progesterone. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, the FTE organoids express one or more of: TUBB4A, FOXJ1, and PAX8.

Also described herein is a quantity of fallopian tube epithelium (FTE) organoids made by a method including generating fallopian tube epithelium (FTE) organoids, providing a quantity of intermediate mesoderm (IM) cells, culturing the IM cells in a matrix and in the presence of at least one first growth factor, and at least first one kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one second growth factor into FTE organoids. In other embodiments, the at least one first growth factor includes WNT4 and at least one kinase inhibitor includes ROCK inhibitor Y-27632. In other embodiments, culturing the IM cells includes about 2 days. In other embodiments, the at least one second growth factor includes follistatin estrogen and progesterone. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, the FTE organoids express one or more of: TUBB4A, FOXJ1, and PAX8.

Further described herein is a composition of fallopian tube epithelium (FTE) organoids. Also described herein is a pharmaceutical composition, including a composition of FTE organoids and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Differentiation of Human iPSC into Intermediate Mesoderm-Like Cells. a) Schematic of developmental stages from inner cell mass to female reproductive tract and fallopian tube. b) Timeline and factors involved in the differentiation of iPSCs into intermediate mesoderm, c) Expression kinetics of mesoderm markers MIXL1- and BRACHYURY during the 6-day differentiation course using Mae et. al., protocol. Mae, S. et al. Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells. Nat Commun 4, 1367, doi:10.1038/ncomms2378 (2013). d) Immunocytochemistry demonstrating expression and localization of mesoderm marker BRACHYURY during differentiation. e) Expression of intermediate mesoderm markers, PAX2, OSR1, GATA3 and WT1 during the 6-day differentiation course. f) Pluripotency markers POU5F1, SOX2 and TDGF1 expression during the 6-day differentiation course. Relative gene expression to iPSC stage (Day 0) was calculated using □□Ct method and normalized endogenous GAPDH level for 87iCTR-n3 iPSC line. Error bars are Standard Error of the Mean (SEM) (n=3 independent experiments). ANOVA with Tukey post-hoc test was used for this analysis, with significance at *p≤0.05, **p≤0.01, ****p≤0.0001.

FIG. 2: Differentiation of Intermediate Mesoderm into Fallopian Tube Epithelium Cell. a) Timeline and factors involved in the differentiation of intermediate mesoderm into fallopian tube epithelium precursor cells. b) qPCR quantification of gene expression kinetics for Müllerian duct markers. WT1 and OVGP1, throughout Müllerian duct differentiation. c) Immunocytochemistry demonstrating protein production and localization of WT1 during Müllerian duct differentiation. d) mRNA fold change comparison of kidney markers SIX2 and FOXD1 in response to Müllerian (WNT4, black bars) vs nephric duct (WNT3a, grey bars) differentiations. e) qPCR quantification of gene expression kinetics for Müllerian duct markers, WT1 and OVGP1, throughout the nephric duct differentiation. Relative gene expression to iPSC stage (Day 0) was calculated using ΔΔCt method and normalized endogenous GAPDH level for 87iCTR-n3 iPSC line. Error bars are SEM (n=3 independent experiments). ANOVA with Tukey post-hoc test was used for this analysis, with significance at *p≤0.05, ***p≤0.001, ****p≤0.0001.

FIG. 3: Progressive Maturation of Fallopian Tube Organoid Over Time in Culture. a) Schematic of factors involved in the differentiation of fallopian tube organoids. b) Bright field image and H&E staining of FTE organoid at day 14. c-e) Immunocytochemistry for FTE markers TUBB4A, FOXJ1 and PAX8 and epithelial marker CDH1 (E-Cadherin) at organoid culture day 14. f) Immunocytochemistry for FTE markers PAX8, TUBB4A, OVGP1 and epithelial marker CDH1 at FTE organoid culture day 45, along with human fallopian tube tissue. g) Gene expression of fallopian tube markers OVGP1 (for 2 different primers), FOXJ1, and TNFaIP2, as well as kidney markers SALL1 and FOXD1 at organoids culture day 45, human fallopian tube and kidney. The color matrix of the heat map represents the log 2(Ratio) of individual gene relative to its expression in iPSC stage. Relative gene expression to iPSC stage (day 0) was calculated using ΔΔCt method and normalized endogenous GAPDH level for 87iCTR-n3 iPSC line. h) H&E staining of FTE organoid at culture day 45 and human fallopian tube tissue.

FIG. 4: Characterization of iPSC lines. All characterized iPSC lines (87iCTR-n3, 01iMEC-n4 and 14iCTR-n6) express pluripotent stem cell markers a) Alkaline Phosphatase (AP) and b) for OCT4, NANOG, SOX2, TRA160, TRA181, SSEA4 based on immunocytochemistry, as well as c) exhibit a normal karyotype based on G-band analysis.

FIG. 5: IM differentiation efficiency for established protocols. Expression kinetics of mesoderm markers a) MIXL1- and b) BRACHYURY during the 6-day differentiation course of iPSC lines 87iCTR-n3, 01iMEC-n4 and 14iCTR-n6 using protocols from Xia, Y. et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol 15, 1507-1515, doi:10.1038/ncb2872 (2013)., Mae et. al. (2013), Takasato, M. et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 16, 118-126, doi:10.1038/ncb2894 (2014).

FIG. 6: Substitution to Mae et al. protocol with BMP4 from BMP7. a) Expression of intermediate mesoderm marker, PAX2, during the 6-day differentiation course of iPSC lines 87iCTR-n3, 01iMEC-n4 and 14iCTR-n6 using protocols from Xia et. al. (2013) and Mae et. al. (2013). b) Expression kinetics of mesoderm markers MIXL1- and BRACHYURY during differentiation with BMP4 vs BMP7 treatment for 87iCTR-n3 iPSC line. c) Expression kinetics of ectoderm marker NCAM and endoderm marker SOX17 during differentiation with BMP4 vs BMP7 treatment for 87iCTR-n3 iPSC line.

FIG. 7: Immunocytochemistry analysis throughout the Müllerian duct differentiation for production of a) gut marker CDX2 and b) lung marker NKX2.1.

FIG. 8: Expression kinetics of a,b) early mesonephric duct markers HOXB7 and GATA3, c,d) Müllerian duct markers WT1 and OVGP1 and e,f) kidney markers SIX2 and FOXD1 with WNT4/CHIR99021 treatment of the 87iCTR-n3 iPSC line.

FIG. 9: Bright field image of 3D structure of FTE organoid at day 14 in a) Matrigel with Phenol Red vs b) Matrigel without Phenol Red.

FIG. 10: Immunocytochemistry analysis of day 21 organoid for kidney marker SIX2 with a) WNT3A treatment as a positive control for kidney differentiation compared with b) WNT4 treatment for Müllerian duct differentiation.

FIG. 11: PAX8 expression pattern during the course of differentiation: a) qPCR quantification of gene expression kinetics for PAX8 in response to Müllerian (WNT4, black bars) vs nephric duct (WNT3a, gray bars) differentiation. b) Immunocytochemistry demonstrating PAX8 expression during Müllerian duct differentiation. c) Immunocytochemistry demonstrating protein production and localization of PAX8 during nephric duct differentiation. d) Immunocytochemistry for PAX8 at FTE organoid culture day, day 30 and human fallopian tube tissue. e) Gene expression of PAX8 at organoids culture day 45, human fallopian tube and kidney. The color matrix of the heat map represents the log 2(Ratio) of PAX8 to its expression at the iPSC stage. Relative gene expression to iPSC stage (day 0) was calculated using DDCt method and normalized to endogenous GAPDH level for 87iCTR-n3 iPSC line.

FIG. 12: Bright field image of Müllerian duct differentiation in monolayer culture for 6 days. Red arrow indicates the epithelial bud formation at day 4.

FIG. 13: a) Bright field image of 3D structure of FTE organoid from 01iMEC-n4 iPSC lines at day 30 in. b) H&E staining of FTE organoid from 01iMEC-n4 iPSC lines at day 30. (c-e) Immunocytochemistry for FTE markers TUBB4A and PAX8, and epithelial marker CDH1 (E-Cadherin) at organoid culture day 30, 87CTR-n3 iPSC, 01iMEC-n4 iPSC biological replicate1 and 01iMEC-n4 iPSC biological replicate2 respectively

FIG. 14: Development and Characterization of an iPSC-Derived Fallopian Tube Organoid. Immunocytochemistry for FTE markers TUBB4A and OVGP1 at FTE organoid culture day 45, along with human fallopian tube tissue in high magnification.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22^nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3^rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

As described, it is now widely-understood that a majority of high-grade serous carcinomas (HGSC) arise from the secretory cells of the fallopian tube epithelium (FTE). What is needed to build upon these discoveries is relevant in vitro human models that can recapitulate tissue-specific architecture and study early alterations that lead to FTE transformation as well as the initiation and progression of HGSC.

Fallopian tube epithelium (FTE) is composed of a polarized columnar epithelium with multiple cell types including ciliated and secretory cells. Current fallopian tube models, including ex vivo and three-dimensional (3D) spheroid models, demonstrate the importance of the polarity of cells in recreating secretory and ciliated cells. However, FTE cells in these models have a reduced proliferation rate due to induced senescence because of the lack of convoluted luminal architecture and the ectopic microenvironment. Mouse models, including patient-derived xenograft and genetically engineered mice, have overcome some of these limitations and yielded significant insights into the basis of cancer development. However, the complexity of a human tumor is not reliably represented in mouse models. Furthermore, it has been a challenge to engineer silent and expressed mutations with the correct expression timeframe as well as accurate targeting to specific tissue and cell types, such as the secretory cell of the fallopian tube. While biopsy derived 3D human fallopian tube organoid models exists, such approaches are dependent on human biopsy and fallopian tissue with high-risk mutations including BRCA1 cannot be collected for research purposes.

Alternatively, induced pluripotent stem cell (iPSC) technology and 3D-tissue engineering provide powerful tools to recapitulate physiologically relevant aspects of disease progression in vitro and to enhance monolayer cell culture studies. Induced PSCs are generated by reprogramming somatic cells and can be subsequently differentiated into most any cell type. Recently, patient-derived iPSCs have been used to model several inherited human diseases and to successfully generate relevant cell types that displayed disease pathogenesis. However, remaining challenges with iPSC-based modeling include establishing direct differentiation protocols for desired cell types and integrating the cells into functional tissue structures.

Here, the Inventors describe a rapid and efficient method to create an iPSC-derived 3D model of human FTE with the desired cell types and luminal architecture. The female reproductive tract, including FTE, arises from the Müllerian duct in parallel to the urinary system from intermediate mesoderm (IM) of the urogenital ridge in the posterior primitive streak. As such, the Inventors have recapitulated Müllerian development in vitro by starting with established IM protocols and then pro-Müllerian growth factors were used to develop FTE precursors. Correct differentiation was monitored through the expression of cell-related markers such as PAX2, GATA3, OSR1, WT1, and OVGP1. Further differentiation to a FTE lineage was obtained on a 3D growth platform, which enabled the FTE organoid to self-organize into a convoluted luminal structure. Importantly, staining of secretory and ciliated cellular components demonstrated that these structures accurately model fallopian tube function. An iPSC-derived fallopian tube organoid model does not require patient biopsy because the Inventors can generate iPSC from blood cells or skin cells even with known genetic mutations.

Therefore, the Inventors believe the iPSC-derived FTE organoid system could be more beneficial and an ideal platform for identifying the molecular mechanisms leading to early pathogenesis and recapitulating ovarian cancer progression in a controlled environment with high risk mutations.

Described herein is a method for generating a fallopian tube epithelium (FTE), including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in the presence of at least one first growth factor and at least one induction molecule to generate mesoderm cells, further culturing the mesoderm cells in the presence of at least one second growth factor, at least one second induction molecule, and at least first one kinase inhibitor to generate intermediate mesoderm (IM) cells, additionally culturing the IM cells in the presence of at least one third growth factor, and at least one second kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one fourth growth factor into FTE. In other embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs). In various embodiments, the hiPSCs are obtained from reprogrammed blood cells, including red blood cells, or white blood cells. In various embodiments, the reprogrammed blood cells are from a subject with a hereditary cancer mutation, such as BRCA1. In various embodiments, the hiPSCs possess one or more cancer mutations. In various embodiments, the induction molecule is a GSK-3 inhibitor. In various embodiments, the kinase inhibitor in a RHO kinase inhibitor. In other embodiments, the at least one first growth factor includes activin A, and the at least one induction molecule includes CHIR99021. In various embodiments, the concentration of activin A is 50-150 ng/ml. In various embodiments, the concentration of activin A is 100 ng/ml. In various embodiments, the concentration of CHIR99021 is 1-5 μM. In various embodiments, the concentration of CHIR99021 is 3 μM. In various embodiments, the hPSCs are cultured in a media including DMEM/F12, Glutamax, 500 U/ml penicillin streptomycin (Gibco) and 2% fetal bovine serum (FBS). In other embodiments, culturing the hPSCs includes about 1-0 days. In other embodiments, culturing the hPSCs includes about 2 days. In other embodiments, the at least one second growth factor includes BMP4, the at least one second induction molecule includes CHIR99021, and the at least one first kinase inhibitor includes ROCK inhibitor Y-27632. In various embodiments, the concentration of BMP4 is 50-150 ng/ml. In various embodiments, the concentration of BMP4 is 100 ng/ml. In various embodiments, the concentration of CHIR99021 is 1-5 μM. In various embodiments, the concentration of CHIR99021 is 3 μM. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 1-30 μM. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 10 μM. In various embodiments, the mesoderm cells are culture in a media including DMEM/F12, Glutamax, supplemented with 0.1 mM non-essential amino acids, 500 u/ml penicillin/streptomycin, 0.55 mM 2-mercaptoethanol, and 10% knock-out serum replacement (KOSR). In other embodiments, further culturing the mesoderm cells includes about 1-3 days. In other embodiments, further culturing the mesoderm cells includes about 2 days. In other embodiments, the at least one third growth factor includes WNT4 and at least one second kinase inhibitor includes ROCK inhibitor Y-27632. In various embodiments, the concentration of WNT4 is 50-150 ng/ml. In various embodiments, the concentration of WNT4 is 100 ng/ml. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 1-30 μM. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 10 μM. In various embodiments, the additionally the IM cells are cultured in DMEM/F12, 500 u/ml penicillin/streptomycin, and 10 ml reconstituted Ultroser G serum substitute. In other embodiments, additionally culturing the IM cells includes about 1-3 days. In other embodiments, additionally culturing the IM cells includes about 2 days. In other embodiments, the at least one fourth growth factor includes follistatin estrogen and progesterone. In various embodiments, the concentration of follistatin is 10-30 ng/ml. In various embodiments, the concentration of follistatin is 20 ng/ml. In various embodiments, the concentration of estrogen is 0.1-5 ng/ml. In various embodiments, the concentration of estrogen is 1 ng/ml. In various embodiments, the concentration of progesterone is 10-60 ng/ml. In various embodiments, the concentration of progesterone is 33 ng/ml. In various embodiments, differentiating Mullerian epithelium cells includes about 2 days. In various embodiments, differentiating Mullerian epithelium cells includes about 2-10, 10-20, 20-30, 40-50, 50-60, or 60-70 days. In other embodiments, the IM cells are organized as a spheroid. In other embodiments, spheroids are subsequently cultured in a matrix. In various embodiments, the matrix is Matrigel. In various embodiments, the matrix includes one or more extracellular matrix proteins. In various embodiments, the matrix includes phenol red. In other embodiments, the FTE are organized as an organoid. In other embodiments, the FTE organoids are matured by further culturing for about 10-20, 20-30, 40-50, 50-60, or 60-70 days. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, the FTE organoids are cultured in a condition media from FTE cells freshly isolated from tissue. In other embodiments, mesoderm cells express one or more of: BRACHYURY and MIXL1. In other embodiments, IM cells express one or more of: PAX2, OSR1, GATA3, and WT1. In other embodiments, Mullerian epithelium cells express one or more of: WT1 and OVGP1. In other embodiments, Mullerian epithelium cells do not express one or more of: SIX2, FOXD1, CDX2 and NKX2-1.

Further described herein is a method of generating fallopian tube epithelium (FTE) organoids, providing a quantity of intermediate mesoderm (IM) cells, culturing the IM cells in a matrix and in the presence of at least one first growth factor, and at least first one kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one second growth factor into FTE organoids. In other embodiments, the at least one first growth factor includes WNT4 and at least one kinase inhibitor includes ROCK inhibitor Y-27632. In various embodiments, the concentration of WNT4 is 50-150 ng/ml. In various embodiments, the concentration of WNT4 is 100 ng/ml. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 1-30 μM. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 10 μM. In other embodiments, culturing the IM cells includes about 2 days. In other embodiments, the at least one second growth factor includes follistatin estrogen and progesterone. In various embodiments, the concentration of follistatin is 10-30 ng/ml. In various embodiments, the concentration of follistatin is 20 ng/ml. In various embodiments, the concentration of estrogen is 0.1-5 ng/ml. In various embodiments, the concentration of estrogen is 1 ng/ml. In various embodiments, the concentration of progesterone is 10-60 ng/ml. In various embodiments, the concentration of progesterone is 33 ng/ml. In various embodiments, differentiating Mullerian epithelium cells includes about 2 days. In various embodiments, differentiating Mullerian epithelium cells includes about 2-10, 10-20, 20-30, 40-50, 50-60, or 60-70 days. In other embodiments, the IM cells are organized as a spheroid. In other embodiments, spheroids are subsequently cultured in a matrix. In various embodiments, the matrix is Matrigel. In various embodiments, the matrix includes one or more extracellular matrix proteins. In various embodiments, the matrix includes phenol red. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, the FTE organoids are cultured in a condition media from FTE cells freshly isolated from tissue. In other embodiments, the FTE organoids express one or more of: TUBB4A, FOXJ1, PAX8 and CDH1. In various embodiments, the organoids include ciliary, luminal and/or secretory structures.

Also described herein is a quantity of fallopian tube epithelium (FTE) organoids made by a method including generating fallopian tube epithelium (FTE) organoids, providing a quantity of intermediate mesoderm (IM) cells, culturing the IM cells in a matrix and in the presence of at least one first growth factor, and at least first one kinase inhibitor to generate Mullerian epithelium cells, and differentiating Mullerian epithelium cells by addition of at least one second growth factor into FTE organoids. In other embodiments, the at least one first growth factor includes WNT4 and at least one kinase inhibitor includes ROCK inhibitor Y-27632. In various embodiments, the concentration of WNT4 is 50-150 ng/ml. In various embodiments, the concentration of WNT4 is 100 ng/ml. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 1-30 μM. In various embodiments, the concentration of ROCK inhibitor Y-27632 is 10 μM. In other embodiments, culturing the IM cells includes about 2 days. In other embodiments, the at least one second growth factor includes follistatin estrogen and progesterone. In various embodiments, the concentration of follistatin is 10-30 ng/ml. In various embodiments, the concentration of follistatin is 20 ng/ml. In various embodiments, the concentration of estrogen is 0.1-5 ng/ml. In various embodiments, the concentration of estrogen is 1 ng/ml. In various embodiments, the concentration of progesterone is 10-60 ng/ml. In various embodiments, the concentration of progesterone is 33 ng/ml. In various embodiments, differentiating Mullerian epithelium cells includes about 2 days. In various embodiments, differentiating Mullerian epithelium cells includes about 2-10, 10-20, 20-30, 40-50, 50-60, or 60-70 days. In other embodiments, the IM cells are organized as a spheroid. In other embodiments, spheroids are subsequently cultured in a matrix. In various embodiments, the matrix is Matrigel. In various embodiments, the matrix includes one or more extracellular matrix proteins. In various embodiments, the matrix includes phenol red. In other embodiments, the FTE organoids are matured by further culturing for about 14-65 days. In other embodiments, the FTE organoids are cultured in a condition media from FTE cells freshly isolated from tissue. In other embodiments, the FTE organoids express one or more of: TUBB4A, FOXJ1, PAX8 and CDH1. In various embodiments, the organoids include ciliary, luminal and/or secretory structures.

Further described herein is a composition of fallopian tube epithelium (FTE) organoids. Also described herein is a pharmaceutical composition, including a composition of FTE organoids and a pharmaceutically acceptable carrier. In various embodiments, the FTE organoids are derived from human pluripotent stem cells, including are human induced pluripotent stem cells (hiPSCs). In various embodiments, the hiPSCs are obtained from reprogrammed blood cells, including red blood cells, or white blood cells. In various embodiments, the reprogrammed blood cells are from a subject with a hereditary cancer mutation, such as BRCA1. In various embodiments, the hiPSCs possess one or more cancer mutations.

Also described herein is a method including selecting a subject in need of treatment for a reproductive tract disease and/or condition, administering to the subject a quantity of fallopian tube epithelium (FTE) cells, wherein the FTE cells are capable of treating the reproductive tract disease and/or condition. In various embodiments the reproductive tract disease includes the upper reproductive tract, including fallopian tubes, ovary and uterus. In various embodiments, the reproductive tract disease and/or condition includes damage and/or dysfunction in the reproductive tract. This includes for example, damage and/or dysfunction associated with endometriosis, uterine fibroids, pelvic inflammatory disease, polyps, scarring, unusual shape. In various embodiments, the treatment of the reproductive tract disease includes FTE cells repairing, regenerating cellular structures in the reproductive tract, and/or restoring functional capabilities of existing cells in the reproductive tract.

EXAMPLE 1 iPSC Culture

Human B-cells, mammary epithelial cells and fibroblasts were used to derive iPSCs, which were cultured in mTeSR®1 medium (STEMCELL) on growth factor-reduced Matrigel™ Matrix (BD Biosciences)-coated plates at 37° C. in a 5% CO₂ incubator. Briefly, 70-90% confluent human iPSC colonies were dissected into small squares using the EZ Passage tool (Invitrogen). For weekly passaging, colonies were lifted carefully with a cell scraper, removed using a 5 ml glass pipette, and replated at a 1:6 ratio.

EXAMPLE 2 Directed Differentiation of iPSCs in Chemically Defined Conditions

Human iPSCs were split onto Matrigel-coated plates, and cultured in mTeSR®1 medium until 80% confluent. Three intermediate protocols were compared, according to Mae et al. (2013), Xia et al. (2013), and Takasato et al. (2014). The final protocol that achieved best efficiency and results for creating fallopian tube epithelium was a modified Mae et. al., (2013) protocol, as defined below.

• • Day 0-2: Cells were exposed to 100 ng/ml human recombinant activin A (Stemgent) and 3 μM CHIR99021 (Cayman Chemicals) to differentiate towards mesoderm, and cultured in DMEM/F12 (Gibco)+Glutamax (Invitrogen) supplemented with 500 U/ml penicillin streptomycin (Gibco) and 2% fetal bovine serum (FBS) with addition of 10 μM ROCK inhibitor Y-27632 (Stemgent).
  • Day 2-4: To differentiate towards IM, media was changed to DMEM/F12 (Gibco)+Glutamax (Invitrogen) supplemented with 0.1 mM non-essential amino acids (Invitrogen), 500 u/ml penicillin/streptomycin (Gibco), 0.55 mM 2-mercaptoethanol, 10% KOSR (Invitrogen), 100 ng/ml BMP4 (R&D Systems), 3 μM CHIR99021 (Cayman Chemicals), and 10 μM ROCK inhibitor Y-27632 (Stemgent).
  • Day 4-6: Spheroids were collected from wells and re-plated per methods described below. To differentiate the spheroid cultures towards Müllerian epithelium, media was changed to Fallopian tube media (FTM) containing DMEM/F12 (Gibco)+500 u/ml penicillin/streptomycin (Gibco), and 10 ml reconstituted Ultroser G (15950-017, Pall) and 10 μM ROCK inhibitor Y-27632 (Stemgent). To the FTM, 100 ng/ml human recombinant WNT4 (R&D Systems) with or without 3 μM CHIR99021(Cayman Chemicals), 100 ng/ml human recombinant WNT3A (R&D Systems) with or without 3 μM CHIR99021 (Cayman Chemicals) were added.
  • Day 6-8: FTM was changed and 20 ng/ml human recombinant Follistatin (Peprotech), 1 ng/ml estrogen and 33 ng/ml progestrone were added.

EXAMPLE 3 Growing FTE Organoids from Spheroids in Matrigel

Spheroids were collected on day 4 from every well under a stereomicroscope using a 200 μl barrier pipette tip and pooled into a 1.5 ml microcenterfuge tube. Spheroids were then mixed with 50 μl Matrigel (BD Biosciences) containing estrogen (1 ng/ml) and progesterone (33 ng/ml), and slowly pipetted into the middle of one well of a 24-well Nunclon delta surface dish. The 3D droplet was allowed to solidify for 10-15 minutes in a tissue incubator, and Matrigel beads were then bathed in FTM supplemented with the same concentration of growth factors. Media was replaced every 3-4 days as necessary and cells were replated every two weeks.

EXAMPLE 4 RNA Isolation and Real-Time PCR Analysis

Total cellular RNA was isolated using Qiagen RNeasy Mini kit according to the manufacturer's recommendations (Qiagen). Of RNeasy-treated total RNA (1 μg) was used for cDNA synthesis using the Quantitect Reverse Transcription Kit for cDNA synthesis for PCR (Qiagen). Real-time PCR was performed using the SYBR Green Supermix (BioRad). The levels of expression of respective genes were normalized to corresponding GAPDH values and are shown as fold change relative to the value of the control sample. All sample analyses were carried out in triplicate. The list of primers used for real-time PCR experiments are listed in Table 1.

TABLE 1
RT-PCR PRimers

Primer	Forward	Reverse
BRACHYURY	GCTGTGACAGGTACCCAACC	CATGCAGGTGAGTTGTCAGAA
	[SEQ ID NO: 1]	[SEQ ID NO: 2]
FOXD1	GACTCTGCACCAAGGGACTG	CAATTGGAAATCCTAGCAGTAAAGT
	[SEQ ID NO: 3]	[SEQ ID NO: 4]
FOXJ1	GGGGTGGGAGCAACTTCT	CCTCCTCCGAATAAGTATGTGGT
	[SEQ ID NO: 5]	[SEQ ID NO: 6]
GAPDH	GTGGACCTGACCTGCCGTCT	GGAGGAGTGGGTGTCGCTGT
	[SEQ ID NO: 7]	[SEQ ID NO: 8]
GATA3	CTCATTAAGCCCAAGCGAAG	GTCTGACAGTTCGCACAGGA
	[SEQ ID NO: 9]	[SEQ ID NO: 10]
HOXB7	CCGAGAGTAACTTCCGGATCTA	CGTCAGGTAGCGATTGTAGTGA
	[SEQ ID NO: 11]	[SEQ ID NO: 12]
MIXL1	GGTACCCCGACATCCACTT	GCCTGTTCTGGAACCATACCT
	[SEQ ID NO: 13]	[SEQ ID NO: 14]
NCAM	GATTCCTCCTCCACCCTCAC	CAATATTCTGCCTGGCCTGG
	[SEQ ID NO: 15]	[SEQ ID NO: 16]
NKX2.1	TCATTTGTTGGCGACTGG	TGCTTTGGACTCATCGACAT
	[SEQ ID NO: 17]	[SEQ ID NO: 18]
OSR1	GGACCTCTGCGGAACAAG	TGCAGGGAAGGGTGGATA
	[SEQ ID NO: 19]	[SEQ ID NO: 20]
OVGP1	AAGCTGTTGCTGTGGGTTG	TGTGCCCAGTTGGTGAAAT
	[SEQ ID NO: 21]	[SEQ ID NO: 22]
OVGP1-2	AATTCTCTACCCAGAGTTCAACAA	CCGATGGACAGTAGTGTTTTCA
	A [SEQ ID NO: 23]	[SEQ ID NO: 24]
PAX2	GAAGTGCCCCCTTGTGTG	TCGTTGTAGGCCGTGTACTG
	[SEQ ID NO: 25]	[SEQ ID NO: 26]
POU5F1	ACCCACACTGCAGCAGATCA	CCACACTCGGACCACATCC
	[SEQ ID NO: 27]	[SEQ ID NO:28]
SALL1	ATTGCAGCCTAGCCAAAAAG	ACCAGCTGAGCAGAAAGGTC
	[SEQ ID NO: 29]	[SEQ ID NO: 30]
SIX2	CAGGTCAGCAACTGGTTCAA	AGCTGCCTAACACCGACTTG
	[SEQ ID NO: 31]	[SEQ ID NO: 32]
SOX17	ACGCCGAGTTGAGCAAGA	TCTGCCTCCTCCACGAAG
	[SEQ ID NO: 33]	[SEQ ID NO: 34]
SOX2	GGGGGAATGGACCTTGTATAG	GCAAAGCTCCTACCGTACCA
	[SEQ ID NO: 35]	[SEQ ID NO: 36]
TDGF1	AGGGAACAATGACAGAGTGTGA	CCC GAGATGGACGAGCAAAT
	[SEQ ID NO: 37]	[SEQ ID NO: 38]
TNFaIP2	GAGCCACGGCTTTGACAC	GTGCGTGAACCTCTTGAACA
	[SEQ ID NO: 39]	[SEQ ID NO: 40]
WT1	GAATGCATGACCTGGAATCA	TCTGCCCTTCTGTCCATTTC
	[SEQ ID NO: 41]	[SEQ ID NO: 42]

TABLE 2
Cell Background Information.

Cell ID	Parent Tissue	Biopsy	Gender	Karyotype
87iCTR-n3	EBV B-cell	Blood	female	normal
01iMEC-n4	Mammary	Mammary	female	normal
	epithelial cells	epithelial
14iCTR-n6	Fibroblast	Skin	female	normal

EXAMPLE 5 Immunocytochemistry for Monolyer Culture

Monolayer cultures were grown on poly-1-lysine and ornithine coated glass coverslips. Cells were washed once with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in 1× PBS for 20 minutes and permeabilized in PBS containing 0.5% Triton X-100 (PBS-T) (Sigma) for 5 minutes at room temperature. Cells were then, blocked with 10% FBS in PBS for 1 hour at room temperature, and followed by 2 hour incubation in primary antibodies (see below section). The cultures were washed with PBS-T three times for 15 minutes each at room temperature and incubated with species-specific AF488 or AF594-conjugated secondary antibodies followed by the nuclei counterstain with DAPI. Following three washes in PBS-T, the coverslips were mounted onto glass slides and were imaged using Nikon/Leica microscopes. Each selected image is representative of a minimum of three independent experiments with at least two technical duplicates.

EXAMPLE 6 Immunocytochemistry for Organoid and Fallopian Tube Tissue

Organoids and fallopian tube tissues were fixed with 4% PFA in 1× PBS for 20 minutes, followed by three PBS washes. The fixed organoids and fallopian tube tissues were then sunk in 30% sucrose at 4° C. overnight and then embedded into OCT (Tissue-Tek). Frozen sections were collected at 12 μm using a cryostat onto glass slides and stored at −80° C. Each section was rehydrated with 1× PBS for 5 min and blocked in a solution of 10% FBS in PBS+0.05% Triton X-100 (PBS-T) for 1 hour at room temperature, followed by 2 hour incubation at room temperature in primary antibodies (see below section) in blocking solution. The slides were washed with PBS-T three times for 15 minutes each at room temperature and incubated with species-specific AF488 or AF594-conjugated secondary antibodies followed by DAPI counterstain. Following three washes in PBS-T, the tissue was covered with a glass slide and imaged using Nikon/Lecia microscopes. Each image selected for figures of this manuscript is representative of a minimum of three independent experiments with at least two technical duplicates.

EXAMPLE 7 Antibodies

The following primary antibodies were used for fluorescence microscopy experiments at 1:200 dilution and secondary antibodies at 1:400 dilution: WT1 (Abcam, ab89901), PAX8 (Proteintech, 21384-1-AP), TUBB4A (Abcam, ab1315), BRACHYURY (Abcam, ab20680), POU5F1 (Stemgent, 09-0023), Nanog (Stemgent, 09-0020), SOX2 (Stemgent, 09-0024), TRA-1-60 (Stemgent, 09-0010), TRA-1-81 (Stemgent, 09-0011), SSEA4 (Stemgent, 09-0006), CDX2 (Biocare Medical, CM226A), SIX2 (Proteintech, 11562-1-AP), FOXJ1 (Abcam, ab40869), CDH1 (R&D System, AF648), OVGP1 (SIGMA, HPA062205) and DAPI (Molecular Probes, D3571).

EXAMPLE 8 Fallopian Tube Tissue Collection

Fallopian tubes were collected from patients undergoing surgery for benign gynecological indications, such as adnexal mass, fibroids, or other conditions not affecting the fallopian tubes. Tissue collection was under IRB PRO00033469. Tissues were inspected and confirmed by pathology to be healthy and not associated with gynecologic malignancy. Upon surgical excision, fallopian tubes were collected in warmed sterile FTM as defined above. Fallopian tubes were rinsed 2 times for 10 mins with Red Blood Cell (RBC) lysis buffer (0.144M NH₄Cl and 0.014M NH₄HCO₃ in 10:1 ratio).

EXAMPLE 9 Statistical Analysis

Statistical analyses were performed by using Prism software (GraphPad Software, La Jolla, Calif.). All quantitative data were expressed as mean values±Standard Error of the Mean (SEM) and analyzed by analysis of variance (ANOVA) followed by a Tukey post-hoc analysis of mean differences in three biological replicates. Differences were considered significant at *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

EXAMPLE 10 Robust Differentiation of Human iPSCs into Intermediate Mesoderm-Like Cells

The mesoderm differentiates into the IM, in a sequential manner determined by the differential expression of specific transcription factors in response to signaling pathways, including WNT, Nodal/Activin and Bone Morphogenetic Proteins (BMPs) (FIG. 1a, b ). The IM subsequently develops in parallel into both the FTE and kidney. At 6 weeks post-fertilization, mesonephric ducts are formed and develop separately into the Müllerian ducts. There are several protocols for kidney development, which primarily utilize signaling molecules in various temporal combinations (FIG. 1b ). However, no successful protocol exists, to date, for iPSC development into fallopian tube.

In order to create a specific 3D iPSC-derived model of FTE, the Inventors used three different human iPSC lines (87iCTR-n3, 01iMEC-n4, 14iCTR-n6) that were reprogrammed initially from B-cells, mammary epithelial cells and fibroblasts, respectively. Each line demonstrated the molecular hallmarks of pluripotent cells, including morphology, normal karyotype, alkaline phosphatase expression and pluripotency markers (FIG. 4), and they were able to differentiate into all three embryonic germ layers (data not shown). The ideal IM differentiation platform was first established by investigating three published protocols for directed differentiation of iPSCs into kidney progenitor cells^21-23. To determine the efficiency of mesoderm induction, PCR and immunocytochemistry were used to assess early mesoderm specific transcription factors MIXL1 and BRACHYURY. A protocol with 100 ng/ml of activin A and 3 μM of CHIR99021 rapidly and efficiently specified human iPSCs (line 87iCTR-n3) into mesoderm, which recapitulates Mae et al (2013) (FIG. 1c ). All three human iPSC lines (87iCTR-n3, 01iMEC-n4, 14iCTR-n6) showed a similar pattern, though the differentiation potential varied slightly between lines (FIG. 5). The peak of BRACHYURY and MIXL1 expression at day 2 followed by subsequent downregulation is consistent with the transient expression of these genes during gastrulation. In addition, BRACHYURY protein production showed a similar kinetic pattern during cell differentiation (FIG. 1d ).

The BMP signaling pathway, known to regulate mesodermal cell determination, was next examined to further specify mesoderm differentiation into IM. While the Mae et al (2013) protocol uses BMP7, results here showed that exposure to 100 ng/ml of BMP4 provided optimal mesoderm differentiation into IM, based on expression levels of early IM markers PAX2, OSR1 and GATA3 (FIG. 1e ). Once again, all three human iPSC lines showed a similar pattern of differentiation based on PAX2 expression, with slightly varying levels of differentiation between lines (FIG. 6a ). As in FIG. 1c , BMP4 provided the expected transient upregulation of BRACHYURY and MIXL1 seen during gastrulation, which was not recapitulated with BMP7 treatment (FIG. 6b ). Importantly, BMP4 has been shown to regulate early ovarian follicle development and the creation of neonatal mouse uterine epithelium. Notably, BMP4-induced IM was more prone to differentiate towards FTE compared to BMP7, as suggested by the intermediate upregulation of markers primed for the female reproductive tract, namely WT1 (FIG. 1e ). In parallel to mesoderm and IM gene induction, the expression of pluripotency genes POU5F1, SOX2 and TDGF1 was reduced (FIG. 1f ). This new differentiation protocol that included BMP4 appears to be mostly specific for mesoderm, as there was no to very low detectable expression levels for the endoderm-marker SOX17 and the ectoderm-marker NCAM (FIG. 6c ).

Collectively, these data show that human iPSCs can be differentiated into mesoderm and IM. Critically, compared to other tested protocols, this newly developed protocol with BMP4 yielded the highest level of mesoderm and IM differentiation.

EXAMPLE 11 Differentiation of Intermediate Mesoderm into Fallopian Tube Epithelial

Female tract differentiation requires WNT4 for initiation and elongation phases of Müllerian duct formation and subsequently requires the downstream component, Follistatin, to form the Müllerian duct. In order to differentiate IM into FTE, cultures were treated with 100 ng/ml of WNT4 on day 4 followed by 20 ng/ml of Follistatin on day 6 (FIG. 2a ). Müllerian duct formation was confirmed by both PCR and immunocytochemistry, which showed upregulated expression over time of the fallopian tube precursor markers WT1 and OVGP1 (FIG. 2b-c ). Importantly, this protocol was specific to Müllerian duct differentiation, given the lack of expression of the kidney markers SIX2 and FOXD1 (FIG. 2d , black bars), as well as the intestinal and lung markers CDX2 and NKX2-1, respectively (FIG. 7a,b ). Interestingly, WNT4 along with prolonged CHIR99021 exposure led to the expression of early mesonephric duct markers HOXB7 and GATA3 (FIG. 8a,b ). However, compared to WNT4 alone (FIG. 2b ), FTE markers WT1 and OVGP1 did not show increased expression (FIG. 8c,d ). Additionally, as with WNT4 alone (FIG. 2d ), WNT4/CHIR did not induce expression of kidney progenitor markers SIX2 and FOXD1 (FIG. 8e,f ). This suggests that continued CHIR99021 during WNT4 treatment might induce other WNT signaling factors that actually disrupt further Müllerian development.

Since Müllerian duct development occurs in parallel to nephric duct, the kinetics of kidney progenitor formation using the Inventors' new differentiation protocol were also characterized. Treating cultures with WNT3A led to increased expression of the renal marker SIX2 and FOXD1, suggesting the formation of the nephric duct and uteric bud and recapitulating published protocols (FIG. 2d , grey bars). Importantly, this protocol was specific to Müllerian duct differentiation, demonstrated by the lack of expression of the early kidney markers SIX2, FOXD1, and PAX8 (FIG. 2d , FIG. 11a black bars and FIG. 11b red channel). In contrast, WNT3A treatment did not yield differentiated Müllerian duct, based on the nearly 10-fold decreased levels of WT1 and OVGP1 compared to WNT4 treatment in FIG. 2b (FIG. 2e ). Collectively these results demonstrate that activation of WNT4 followed by Follistatin can selectively differentiate IM into FTE precursor cells.

EXAMPLE 12 Generation and Progressive Maturation of Fallopian Tube Organoid Over Time

Remarkably, BMP4- and CHIR99021-treated cultures underwent morphogenesis that was similar to embryonic development. Between 3-5 days of treatment, flat cell sheets condensed into epithelial buds (FIG. 12). Once harvested, these buds formed spheroids, which were placed into Matrigel beads along with pro-Müllerian growth factors. Since optimal IM formed at day 4, spheroids were collected on days 3 to 6. Day 4 was ideal for collection of spheroids, as cells collected on day 3 were immature and grew with unstable structure in Matrigel and spheriods collected after day 6 did not persist in culture. When these spheroids were grown in Matrigel that contained phenol red, they formed an organoid structure (FIG. 9a ). In contrast, when spheroids were grown in Matrigel not containing phenol red, they became branched and formed an unorganized matrix (FIG. 9b ). As phenol red is a weak estrogen mimic, these results indicate the significance of estrogen on FTE differentiation and maturation

While the estrogenic properties of phenol-red initially improved organoid growth and organization, it was unable to sustain the organoids over longer periods. Steroid hormones, and estrogen in particular, are known to regulate development of the female reproductive tract, and estrogen has been shown to mediate cellular proliferation and differentiation during embryogenesis². Therefore, to increase the architectural complexity and gain the structure of the plicae, fallopian tube organoids were exposed to estrogen (E2) and progesterone (P4) (FIG. 3a ). In addition, conditioned media from FTE cells freshly isolated from patient tissue was used to provide other factors in the fallopian tube milieu that may be necessary for fallopian tube development.

This optimized protocol provided long-term organoids in matrigel that displayed luminal structures (FIG. 3a,b ). Immunocytochemistry demonstrated that these fallopian tube structures contained ciliated (TUBB4A and FOXJ1) and secretory (PAX8) cellular components (FIG. 3c-e ). However, these markers were found throughout the organoid, instead of in specific fallopian tube compartments as would be expected in mature FTE. In addition, there was no expression of the mature epithelial cell marker CHD1 (FIG. 3e ). Also, immunocytochemistry confirmed the lack of expression of kidney marker SIX2, compared to WNT3a treatment as a positive control for kidney differentiation (FIG. 10a,b ) Collectively, this suggests that while FTE precursor cells were detected in day 14 organoids, functional maturation of FTE required two months, similar to the timeline for human development.

To obtain functional maturation of FTE, the iPSC-derived FTE organoids were extended in culture in 3D Matrigel for an extended period with estrogen and progesterone supplemented media. Immunocytochemistry at day 30 showed that secretory cell marker PAX8 and ciliated cell marker TUBB4A, as well as the epithelial cell marker CDH1, exhibited similar expression patterns in FTE organoids model from two different iPSC lines (FIG. 13), Results at 45 days showed that the FTE cil ated marker, TUBB4A, and secretory cell markers, OVGP1 and PAX8 and the epithelial cell marker, CDH1, were now expressed in the expected sub-compartments seen in mature FTE, with TUBB4A being highly expressed along the lumen (FIG. 3f , FIG. 14). Importantly, the cellular expression and organization was comparable to fresh human fallopian tube tissue (FIG. 3f ). The differentiation of iPSC-derived organoids into fallopian tube mature cells overtime was further demonstrated heat map analysis, which showed increased expression of fallopian tube-specific ciliated markers, FOXJ1 and secretory cell markers, OVGP1 that were similar to human fallopian tube tissue (FIG. 3g ). Critically, iPSC-derived FTE organoids not only expressed both ciliated and secretory cell markers but also they formed visible cilia, further demonstrating that this novel model closely mimics the proper physiology and anatomy of the human FTE (FIG. 3h ).

EXAMPLE 13 Discussion

Human iPSCs are derived using technologies to reprogram adult cells back to a pluripotent state with the subsequent potential to differentiated into any cell type. Reprogramming to human iPSC possesses the essential advantages of eliminating the requirement for embryonic material while allowing for the generation of pluripotent cells with known genetic factor in a patient-specific manner. Moreover, combined with 3D cell culture techniques, 3D-iPSC derived organoid models can be developed which reconstitute features of organs, thereby overcoming many of the limitations of traditional monolayer cell culture systems by mimicking more closely the complex cellular heterogeneity and cell-cell/cell-matrix interactions.

Intermediate mesoderm generates several components of the urogenital system, including reproductive tracts and also the kidneys, the gonads, and their respective duct systems. Priming the IM into only the desired cell fate has been a challenge for the iPSC-derived organoid model. To the Inventors' knowledge, all established iPSC-derived IM platforms have been developed to model the kidney formation using the combination of WNT, Nodal/Activin, and BMP signaling pathways. In this study, different WNT signaling pathways were modulated to successfully develop a novel differentiation protocol with key components to drive Müllerian duct and the female reproductive tract development rather than kidney. The most efficient differentiation platform to mimic embryonic fallopian tube development used CHIR99021 and activin A, along with BMP4 that was discovered to be critical for conversion of IM into female reproductive tract or Müllerian duct. To the Inventors' knowledge, this study presents the first iPSC-derived FTE in vitro model. The Inventors have demonstrated direct differentiation of human iPSCs into a fallopian tube-like precursor cells and, moreover, established iPSC-derived human fallopian tube organoids that recapitulate complexity of 3D architecture of FTE tissue.

During embryogenesis, WNT signaling pathways play a prominent role in regulating cell fate specification and in determining cell polarity and migration. WNT signaling is often correlated with cell proliferation and tissue repair after acute injury, rather than with differentiation of stem cells into adult tissues. However, recent human FTE organoid models with bi-potent FTE stem cells indicated the function of WNT3A in fallopian epithelial renewal and the regulation of stemness, suggesting the distinct role of WNT pathways in embryonic development and adult stem cell function. The current study found that activation of WNT4, instead of WNT3A, and activation of the downstream effector Follistatin were vital to specify IM differentiation into Müllerian duct and an FTE cell fate in vitro. Interestingly, the Inventors also showed that subsequent activation of WNT3A rather than WNT4 promoted nephric duct formation, which reflects the different molecular ability of WNTs to regulate specific cell fates.

Many fundamental cellular processes are differentially regulated between 2D and 3D cultures. Exposure of differentiated iPSCs to a 3D growth platform enabled them to self-organize into luminal structures that model fallopian tube function with secretory and ciliated cell components. Critically, the exposure to primary fallopian tube epithelial conditioned media is required for maintenance of 3D FTE luminal structure and architecture, indicating that there are unknown factors responsible for the organization of cellular structure of the fallopian tube.

In this study, the Inventors generated the basis platform for direct differentiation of iPSC into fallopian tube epithelial cell, however there is room for further improvement with regard to long term culturing and functional maturity. The growth and successful cultivation may dependent on different cell-matrix interactions and some other growth factors, which needs to further address.

Induced PSC-based in vitro models of cancer can help to understand pathological processes at the molecular and cellular level. In addition, they provide a critical platform to study drug resistance and to develop novel drug therapies. Importantly for ovarian cancer, iPSC-derived fallopian tube organoids can provide a faithful human cellular model to investigate the fallopian tube origin of serous carcinogenesis in ovarian cancer and to explore early cancer pathogenesis and progression. This platform can also be used study germline mutations that effect ovarian cancer development and to identify the critical steps and the order of genetic alterations involved with high grade serous carcinogenesis. In summary, iPSC-derived fallopian tube epithelium provides a powerful in vitro model of ovarian cancer that can be used to recapitulate early de novo genomic alterations, faithfully model disease progression and ultimately uncover novel treatments.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are fallopian tube epithelium, cells, organoids, and tissue products thereof, methods of generating fallopian tube epithelium, prognostic and/or diagnostic panels that include nucleic acid, peptide and proteins sequences associated with cancers such as ovarian cancer, and the techniques associated with the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.

Claims (19)

The invention claimed is:

1. A method for generating a fallopian tube epithelium (FTE), the method comprising:

providing a quantity of human pluripotent stem cells (hPSCs);

culturing the hPSCs in the presence of at least one first growth factor comprising Activin A and at least one first induction molecule comprising CHIR99021 to generate mesoderm cells;

further culturing the mesoderm cells in the presence of at least one second growth factor comprising BMP4, at least one second induction molecule comprising CHIR99021, and at least one first kinase inhibitor comprising ROCK inhibitor Y-27632 to generate intermediate mesoderm (IM) cells;

additionally culturing the IM cells in the presence of at least one third growth factor comprising WNT4, and at least one second kinase inhibitor comprising ROCK inhibitor Y-27632 to generate Mullerian epithelium cells; and

differentiating the Mullerian epithelium cells by addition of one or more fourth growth factors selected from follistatin, estrogen, or progesterone into FTE.

2. The method of claim 1, wherein the hPSCs are human induced pluripotent stem cells (hiPSCs).

3. The method of claim 1, wherein culturing the hPSCs comprises about 2 days.

4. The method of claim 1, wherein further culturing the mesoderm cells comprises about 2 days.

5. The method of claim 1, wherein additionally culturing the IM cells comprises about 2 days.

6. The method of claim 1, wherein the one or more fourth growth factors comprise follistatin, estrogen, and progesterone.

7. The method of claim 1, wherein the IM cells are organized as spheroids.

8. The method of claim 7, wherein the spheroids are subsequently cultured in a matrix.

9. The method of claim 1, wherein the FTE are organized as organoids.

10. The method of claim 9, wherein the FTE organoids are matured by further culturing for about 14-65 days.

11. The method of claim 1, wherein the mesoderm cells express one or more of: BRACHYURY or MIXL1.

12. The method of claim 1, wherein the IM cells express one or more of: PAX2, OSR1, GATA3, or WT1.

13. The method of claim 1, wherein the Mullerian epithelium cells express one or more of: WT1 or OVGP1.

14. The method of claim 1, wherein the Mullerian epithelium cells do not express one or more of: SIX2, FOXD1, CDX2 or NKX2-1.

15. A method of generating fallopian tube epithelium (FTE) organoids, the method comprising:

providing a quantity of intermediate mesoderm (IM) cells;

culturing the IM cells in a matrix and in the presence of at least one first growth factor comprising WNT4, and at least one kinase inhibitor comprising ROCK inhibitor Y-27632 to generate Mullerian epithelium cells; and

differentiating the Mullerian epithelium cells by addition of one or more second growth factors selected from follistatin, estrogen, or progesterone into FTE organoids.

16. The method of claim 15, wherein culturing the IM cells comprises about 2 days.

17. The method of claim 15, wherein the one or more second growth factors comprise follistatin, estrogen, and progesterone.

18. The method of claim 15, wherein the FTE organoids are matured by further culturing for about 14-65 days.

19. The method of claim 15, wherein the FTE organoids express one or more of: TUBB4A, FOXJ1, or PAX8.

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